The ribosome plays a central role in the process of protein synthesis. The particle is composed of more than 50 macromolecules including proteins and nucleic acids which function synergistically in protein synthesis and in the biogenesis of the particle. The proposed research program is designed to investigate the physical and functional relationship between these components during ribosome assembly and when they are active in protein synthesis. The research will focus on the effects on assembly and function of a recently isolated small polypeptide derived from 30S ribosomal protein S8 (called KS8) which blocks the binding to rRNA of the intact protein from which it was derived. This polypeptide will be used as a new ribosome specific polypeptide "antibiotic". In vitro reconstitution of the 30S subunit in the presence of KS8 will be effected and the protein composition, topology and function of such particles will be investigated. In addition to performing these experiments with conventional in vitro ribosome reconstitution systems, a major effort will be made to develop an in vitro reconstitution procedure which is coupled to transcription of ribosomal RNA. This will provide a new avenue for investigations of the assembly process and an opportunity for detailed investigations of 50S subunit assembly. Following development of the new assembly system investigations of the effect of KS8 on assembly will be conducted.